REverSe TRanscrIptase chain termination (RESTRICT) for selective measurement of nucleotide analogs used in HIV care and prevention

Abstract Sufficient drug concentrations are required for efficacy of antiretroviral drugs used in HIV care and prevention. Measurement of nucleotide analogs, included in most HIV medication regimens, enables monitoring of short‐ and long‐term adherence and the risk of treatment failure. The REverSe TRanscrIptase Chain Termination (RESTRICT) assay rapidly infers the concentration of intracellular nucleotide analogs based on the inhibition of DNA synthesis by HIV reverse transcriptase enzyme. Here, we introduce a probabilistic model for RESTRICT and demonstrate selective measurement of multiple nucleotide analogs using DNA templates designed according to the chemical structure of each drug. We measure clinically relevant concentrations of tenofovir diphosphate, emtricitabine triphosphate, lamivudine triphosphate, and azidothymidine triphosphate with agreement between experiment and theory. RESTRICT represents a new class of activity‐based assays for therapeutic drug monitoring in HIV care and could be extended to other diseases treated with nucleotide analogs.


| INTRODUCTION
The development and availability of antiretroviral drugs has decreased the number of deaths related to Human Immunodeficiency Virus (HIV) by 40% over the past decade. 1 However, sufficient drug concentrations are required to obtain the individual and population-level benefits of antiretroviral therapy (ART) and pre-exposure prophylaxis (PrEP) regimens for HIV treatment and prevention, respectively. [2][3][4][5] In several PrEP trials and implementation studies, concentrations of antiretroviral medications in blood were correlated with PrEP efficacy. 6 Similarly, viral suppression was associated with antiretroviral concentration among people living with HIV receiving ART. 4 Regular monitoring of antiretroviral drug levels and provision of appropriate counseling/feedback could promote ART and PrEP adherence, increase antiretroviral drug concentrations, and improve HIV care. 3 Nucleotide analogs are a suitable target for antiretroviral drug monitoring because of their inclusion in most ART and PrEP regimens 7 and their favorable pharmacokinetics. 8 Nucleotide analogs terminate DNA synthesis by HIV reverse transcriptase (RT) and prevent HIV replication.
Tenofovir diphosphate (TFV-DP), a deoxyadenosine analog, is used in over 80% of ART regimens and in all approved oral PrEP regimens. 7,9 TFV-DP has a 17-day half-life in red blood cells (RBCs) and accumulates 25-fold at steady state compared to the start of drug ingestion, which enables monitoring of long-term medication adherence over a 1 to 3 month period. [10][11][12] Meanwhile, emtricitabine triphosphate (FTC-TP), a deoxycytidine analog, is also included in several ART regimens and in all approved oral PrEP regimens. FTC-TP has a 35-h half-life in RBCs and provides information about recent (1-week) adherence. 13 Measurement of TFV-DP and FTC-TP concentrations can provide information about short-term and long-term adherence and enable investigation of their implications in clinical practice and behavioral science studies. 4,10,11,[13][14][15][16] Liquid chromatography tandem mass spectrometry (LC-MS/MS) is the gold standard for measuring nucleotide analogs, 10,11,17,18 and was used in directly observed therapy trials to determine TFV-DP concentrations corresponding to low (2 doses/week), intermediate (4 doses/week), and high (7 doses/week) PrEP adherence. 10,11 LC-MS/MS was used to establish FTC-TP thresholds corresponding to recent medication ingestion. 10,11,13 Although LC-MS/MS provides accurate and quantitative information about TFV-DP and FTC-TP concentrations, it requires complex sample preparation, highly trained operators, and resource intensive equipment that limit its use to centralized and highly capable laboratories. This hinders routine measurement of antiretroviral concentrations and could delay counseling and interventions in behavioral science studies. 3,19 There are ongoing efforts to miniaturize LC-MS/MS instruments and to integrate the required sample preparation steps into miniaturized cartridges. 20,21,22 However, the per-unit cost and complexity of current miniaturized LC-MS/MS systems still exceeds requirements in low and middle-income settings.
A rapid and inexpensive test that measures nucleotide analogs indicative of long-and short-term adherence in point-of-care (POC) settings like a doctor's office, patient's home, or event setting could be beneficial in monitoring and improving ART and PrEP outcomes. 3,23 Immunoassays and lateral flow assays for measuring antiretroviral drugs were recently developed. [24][25][26][27] Most immunoassays for HIV adherence measurement target tenofovir (TFV), a precursor of TFV-DP that is present in plasma and urine. 12 TFV has a 15-h half-life in plasma and indicates recent ingestion of medication within the prior 48 h. The short half-life of TFV makes its measurement susceptible to conflating recent medication ingestion (≤1 week) with long-term (1-3 months) adherence. We recently developed an immunoassay for measuring clinically relevant concentrations of both TFV and TFV-DP 28 ; however, additional sample preparation is required to separate RBCs from plasma because the antibodies used could not distinguish between TFV and TFV-DP. There is still a strong need for assays that can selectively measure biomarkers of short-and long-term in POC settings. 3,29 We recently developed the REverSe TranscrIptase Chain Termination (RESTRICT) assay for detection of nucleotide analogs based on their enzyme inhibition activity. 30  We use dilution in water as a simple and user-friendly sample preparation mechanism to release intracellular TFV-DP from RBCs and decrease non-specific inhibition by blood matrix components. 31 In a pilot clinical evaluation, RESTRICT measured clinically relevant TFV-DP concentrations in whole blood and identified participants with TFV-DP concentrations corresponding to adequate adherence (≥700 fmol per 3 mm dried blood spot [DBS] punch) among PrEP clients. RESTRICT represents a new class of activity-based diagnostic assays with potential for rapid, POC measurement of antiretroviral drugs used in HIV treatment and prevention.
In this article, we demonstrate that RESTRICT can selectively measure multiple nucleotide analogs based on their chemical structure. Guided by a probabilistic model of RT inhibition, we investigate the impact of assay parameters including nucleotide analog affinity, nucleotide concentration, template length, and template sequence on RESTRICT. We demonstrate selective detection of biomarkers of either long-term or short-term adherence without cross-reactivity using DNA templates that are rich in the endogenous nucleotide that the drug mimics.

| Overview and key assumptions
We present a probabilistic reaction equilibrium model of RESTRICT.
We assume that incorporation of dNTP or nucleotide analog occurs as a random probabilistic event that depends primarily on their relative concentrations. For simplicity, we ignore time-dependent changes in reagent concentration and assume that dNTP and nucleotide analog are available in excess for DNA chain elongation or termination. This model recapitulates experimental results and allows investigation of the role of assay parameters such as nucleotide analog affinity, dNTP concentration, template length, and template sequence on RESTRICT assay performance.
Nucleic acid templates used in the RESTRICT assay consist of a primer binding domain and a chain terminating domain ( Figure 1a). The primer binding domain is located on the 3 0 end of the template and guides primer initiation of DNA synthesis. The chain terminating domain is located directly downstream of the primer binding domain and is designed to enable preferential insertion of specific nucleotide analogs. For example, an assay designed to detect TFV-DP, a deoxyadenosine analog, will use a DNA template rich in thymidine (T) bases to provide ample opportunity for TFV-DP insertion. Although the ideal chain terminating domain for TFV-DP detection is a homopolymeric template consisting of a string of T's, in practice, HIV RT exhibits template preferences and does not efficiently polymerize Poly(dT) templates, 32,33 so the template sequence must be optimized.
There are several contributions to the output fluorescence signal.
The largest signal fraction originates from the interaction between intercalating fluorescent dyes and double stranded DNA (dsDNA) products.
There are smaller magnitude contributions from unpolymerized single stranded DNA (ssDNA) templates and primers. Intercalating dyes provide significantly greater fluorescence when bound to dsDNA; however, they also produce a measurable fluorescence signal when bound to ssDNA ( Figure 1b). For example, PicoGreen™ used in our experiments, provides 11 times greater fluorescence when bound to dsDNA compared to ssDNA. 34,35 The theoretical model also accounts for fluorescence from unpolymerized ssDNA. In the sections below, we provide calculations of the fluorescence contributions of different assay end products.

| Full-length dsDNA products
For a nucleic-acid template with chain terminating domain length, L t , and n bases complementary to the target nucleotide analog (n ≤ L t ), the formation of full-length dsDNA requires n consecutive dNTP insertion events. Assuming these are all independent dNTP insertion events in the presence of excess dNTP and nucleotide analog concentration, the probability of full-length dsDNA formation can be expressed as, where NA ½ is nucleotide analog concentration, dNTP ½ is nucleotide concentration, and K aff is the relative affinity of RT enzyme for the nucleotide analog compared to dNTP.
The fluorescence from full-length dsDNA products, F full , depends on the length of the DNA template and the fluorescence properties of the intercalating dye and can be expressed as,

| Fragment dsDNA products
There is a distribution of dsDNA fragments sizes during RESTRICT assays as previously observed in experimental studies of RT activity. 32 Partial-length dsDNA fragments contribute to the total fluorescence in the RESTRICT assay ( Figure 1b).
where the index i ≥ 1 counts the bases where it is possible to insert nucleotide analog and the maximum possible value of i is the total number of bases in the template, or simply n. P dNTP,iÀ1 is the probability that dNTP molecules were incorporated in the nucleic acid template at all bases preceding the base i where nucleotide analog was inserted and is calculated using Equation (1). P NA is the probability of a single nucleotide analog insertion event and can be expressed as, To determine the total fluorescence contribution from dsDNA fragments, we calculate the sum of fluorescence from all the dsDNA fragments. Adapting Equation (3) and summing the fluorescence from individual dsDNA fragments, the fluorescence from dsDNA fragments can be expressed as, where each termination site i and the resulting dsDNA fragment of length, L i , is known since we know the exact sequence of DNA templates used in the assay.

| Unpolymerized ssDNA
We also account for the background fluorescence contribution of intercalating dye interacting with unpolymerized nucleic acid template. At high nucleotide analog concentrations, very little (if any) dsDNA is formed and most of the fluorescence output comes from unpolymerized ssDNA template ( Figure 1b). In RESTRICT, this background varies with nucleotide analog concentration. Each dsDNA fragment has a corresponding ssDNA fragment with length equal to the difference between the total length of the nucleic acid template and the dsDNA fragment ( Figure 1b). Thus, the background fluorescence from the ssDNA fragments can be calculated as, where L t À L i ½ is the length of the unpolymerized single-stranded portion of a fragment product that was terminated at base i, and the factor D is the fold reduction in fluorescence when intercalating dye is bound to ssDNA rather than dsDNA (D ¼ 11 for PicoGreen™) 34,35 Contributions of bound and unbound primer: DNA templates used in the RESTRICT assay all had a common 20 nt primer binding domain, while the chain terminating domain varied in sequence and length from 45 to 180 nt. Given that the primer binding domain constitutes up to 31% of the total template length, it is important to account for fluorescence that arises from the primer binding domain.
Assuming the reaction goes to completion with all available templates bound to a primer, the fluorescence from dsDNA due to bound primers can be calculated as, where C template is the template concentration and L primer is the primer length.
There is also background fluorescence from unbound primer in solution. To ensure that all templates bound to a primer, we used excess primer in the RESTRICT assay (10 times the template concentration). The fluorescence due to unbound ssDNA primer can be calculated as, where C primer À C t À Á is the difference between primer and template concentration, and the factor D is the fold reduction in fluorescence when intercalating dye is bound to ssDNA rather than dsDNA (D = 11 for PicoGreen). 34,35

| Total fluorescence from RESTRICT assay products
We can calculate the total fluorescence, F total , at the end of the RESTRICT assay by combining Equations (1) to (8), Using this, we can estimate RESTRICT assay performance as we vary assay parameters such as nucleotide concentration, template length, and template sequence.

| Selective detection of nucleotide analogs
We

| Clinical ranges for nucleotide analogs
We estimated clinical ranges, to provide guidelines of concentrations of interest for each nucleotide analog, from published liquid chromatography tandem mass spectrometry measurements. The clinical ranges for FTC-TP, 3TC-TP, and TFV-DP were based on LC-MS/MS measurements in dried blood spots (DBS) (see Table 2

| Data analysis and statistics
We normalized RESTRICT data using fluorescence from negative controls (no RT enzyme) as 0% and fluorescence from positive controls

| Contributions of RESTRICT assay products to endpoint fluorescence
We can account for the contributions of different assay inputs and outputs to the total and normalized fluorescence from the RESTRICT assay. Accounting for background fluorescence from primers and unpolymerized template increases the total fluorescence from the RESTRICT assay (Figure 2a). Our focus in this study was to investigate Effect of nucleotide concentration, template length, and template sequence on detection of TFV-DP using the RESTRICT assay.

| Varying dNTP concentration, template length, and template sequence
We examined the effect of dNTP concentration, template length, and template sequence on RESTRICT assay performance in theory and experiment ( Figure 3). As dNTP concentration increases, we observe less inhibition (and consequently higher fluorescence) as a function of TFV-DP concentrations since higher dNTP concentrations make TFV-DP insertion less likely and more TFV-DP is required to have a similar inhibitory effect (Figure 3a). This has the effect of shifting the inhibition curves to the right. Figure 3b plots the IC 50 value as an exponential function of dNTP concentration and shows good agreement between experimental data and theoretical calculations.  Figure 3d shows an exponential decay in IC 50 value with increasing template length.  Taken together, Figure 3 shows that the probabilistic model for RESTRICT can be used to inform systematic assay design and choose appropriate assay parameters to shift RESTRICT curves to desired concentration ranges for TFV-DP detection. The RESTRICT assay can be shifted towards more sensitive measurement of nucleotide analogs at low concentrations by decreasing the dNTP concentration, increasing the template length, and incorporating a higher fraction of nucleotides that the drug of interest will bind to during polymerization.

| Detection of multiple nucleotide analogs
The RESTRICT assay accounts for the endogenous nucleotide that a drug mimics and Watson-Crick-Franklin base pairing between the template and the drug. We designed DNA templates to enable sensitive detection of nucleotide analogs of interest. Figure 4a shows a RESTRICT curve obtained using 500 nM dNTP, a 180 nt TTCA template, serial dilution dilutions of TFV-DP, and an empirically derived K aff = 0.3. Figure 4b shows a RESTRICT curve obtained using 500 nM dNTP, a 180 nt GGCA template, and serial dilutions of FTC-TP, and empirically derived K aff = 0.2. Figure 4c  F I G U R E 5 Detection of TFV-DP and 3TC-TP using DNA templates designed for selective detection of each drug without cross-reactivity. TFV-DP (A analog) was detected with a thymidinerich TTCA template that excluded G bases to prevent cross-reactivity with 3TC-TP (C analog). Similarly, 3TC-TP was detected with a guanosine-rich GGCA template that excluded T bases. N = 3 clinical sample. For example, TFV-DP and 3TC-TP are common companion drugs used in ART regimens. 7 TFV-DP in red blood cells is a measure of long-term (1-3 month) medication adherence, 10,11 and 3TC-TP in red blood cells indicates recent (<1 week) adherence. 41 Selective measurement of TFV-DP and 3TC-TP is important to avoid conflating recent pill ingestion with long-term adherence.
We designed a guanosine-rich DNA template (180 nt GGAA) for selective detection of 3TC-TP (deoxycytidine analog) and excluded thymidine bases without cross-reactivity with TFV-DP (deoxyadenosine analog). Similarly, we designed a thymidine-rich DNA template that excluded guanosine bases (180 nt TTCA) for selective TFV-DP detection without cross-reactivity with 3TC-TP ( Figure 5). RESTRICT assays with guanosine-rich DNA templates produced the expected inhibition with 3TC-TP, a deoxycytidine analog but did not produce inhibition with TFV-DP even at >1000-fold higher than clinically relevant concentrations (see Table 2). Similarly, the thymidine-rich TTCA template responded only to TFV-DP and did not provide inhibition with 3TC-TP even at concentrations >1000-fold higher than clinically relevant concentrations.

| Benefits of probabilistic model for RESTRICT assays
We developed a probabilistic model that guides the design and optimization of RESTRICT assay parameters including dNTP concentration, template length, and template sequence. Although other models of the

| Reproducibility and potential for quantitative testing
Our goal is to develop a test that is accurate enough to measure clinically relevant nucleotide analog concentrations in whole blood samples. Previous reports suggest that coefficient of variation (CV) less than 15% is suitable for quantitation of antiretroviral drugs in whole blood. 45 The average CV across all our experiments in buffer was <5%, suggesting that RESTRICT assay can achieve the required precision for quantitation of clinical samples. We previously demonstrated that we could achieve a CV of 13.5% when running RESTRICT with whole blood samples diluted in water. 30,31 Improvements in sample preparation-incorporating a heating step after blood dilution to denature blood proteins (e.g., hemoglobin) that nonspecifically inhibit reverse transcriptase 46 -have improved the accuracy even further (data not shown).

| Addressing sources of potential assay interference in clinical samples
We do not anticipate assay interference from endogenous reverse transcriptase (RT) in viremic HIV-infected patients. Studies have estimated the RT activity for patients at very high viremia ($1,000,000 viral copies/mL) to be equivalent to <10,000 fg RT/ml. 47

| Potential for integration into point-of-care format
RESTRICT assays are completed in less than 1 h using readily available nucleic acid analysis reagents and a fluorescence reader. 30 RESTRICT assays are user-friendly and could be integrated into a POC format for use directly at the point of need (e.g., patient's home, doctor's office, or event setting) by using freeze-dried reagents and a low-cost fluorescence reader. DNA amplification reactions that use similar nucleic acid analysis reagents to RESTRICT (i.e., reverse transcriptase, primers, DNA templates, and nucleotides) have been previously integrated into POC formats by our group and others. [49][50][51] We have previously shown that blood dilution to lyse red blood cells is a simple and effective sample preparation strategy to release TFV-DP from red blood cells and minimize non-specific RT inhibition by blood matrix components in whole blood samples from PrEP clients. 31 RESTRICT requires <1 μl of whole blood per test. Each reaction requires addition of 5 μl of 10% whole blood (diluted in water) to 35 μl of reaction mix.

| Deviations between theoretical and experimental RESTRICT
The goal of the theoretical framework is to predict the trends of RESTRICT's fluorescence output and IC 50 as a function of the independent assay design parameters: dNTP concentration, template composition, and length. In this way, the theory can be used to optimize RESTRICT assays by guiding users on how to achieve a desired performance by modifying the template and reaction concentrations. Figure 3 shows that the model can predict the fluorescence and IC50 trends as a function of dNTP concentration, template composition, and length.
The variation in Figure 3b-where there appears to be a growing difference between theory and experiment as dNTP concentration increased-could indicate systematic deviations. For the 1.56 and 6.25 μM data points, experimental data undershoots the theoretical data. This could suggest that at these higher dNTP concentrations (and correspondingly higher template concentrations), additional time might be required to synthesize dsDNA using all available ssDNA templates. We used the same RT enzyme concentration (100 nM) and reaction incubation time (30 min) for all four dNTP concentrations.
Additional assay optimization could ensure that the template is fully polymerized, as predicted in the model, and improve the agreement between the model and experiment. formed on a different template, we hypothesize that the deviation is due to errors in manufacturing the 33.3% template. Alternatively, there have been reports that HIV RT can exhibit DNA sequence preferences. 32,33 Overall, the model predicts the overall trends of RESTRICT fluorescence and IC 50 as a function of the assay parameters and is effective in aiding in the design of RESTRICT assays for measuring specific nucleotide analogs in a particular clinically relevant concentration range.

| Limitations of RESTRICT assay
RESTRICT currently focuses on nucleotide analogs (like TFV-DP, FTC-TP, and 3TC-TP) that accumulate in RBCs which can be lysed for release of the drug by blood dilution in water. Extending RESTRICT to drugs that accumulate primarily in PBMCs rather than RBCs (e.g., AZT-TP) would require additional sample preparation to isolate and lyse PBMCs. This work shows that the model can predict RESTRICT's fluorescence and IC50 trends as a function of the dNTP concentration as well as the template length and composition.
The empirically derived K aff depends on the type (DNA or RNA) and sequence of nucleic acid template, and the choice of RT enzyme used.
If the intent is to use the model to quantitatively predict RESTRICT's output fluorescence, K aff needs to be determined empirically for each template-drug pair.

| CONCLUSIONS
Our results demonstrate that RESTRICT assays can detect multiple nucleotide analog drugs used in HIV treatment and prevention. The activity-based approach to measuring nucleotide analogs in clinical samples presented here could be extended to detect other drugs used in infectious and noncommunicable disease management. Nucleotide analogs and polymerase inhibitors are used to treat hepatitis B, 52 herpes, 53 tuberculosis, 54 cancer, 55,56 and COVID-19. 57 RESTRICT may support therapeutic monitoring and precision dosing of nucleotide analogs for these diseases to ensure efficacy and safety.

ACKNOWLEDGMENTS
We are grateful for helpful conversations with Bob Atkinson, Enos Kline, Tim Cressey, Alex Greninger, Marta Fernandez-Suarez, Jay Rutherford, Rebecca Sandlin, Mehmet Toner, and JaneZhang. We are grateful for experimental support from Emily Blake, Hannah Nguyen, and Nadir Ziane.